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A novel display system is described that allows highly efficient immobilization of heterologous proteins on bacterial surfaces in applications for which the use of genetically modified bacteria is less desirable. This system is based on nonliving and non-genetically modified gram-positive bacterial cells, designated grampositive enhancer matrix (GEM) particles, which are used as substrates to bind externally added heterologous proteins by means of a high-affinity binding domain. This binding domain, the protein anchor (PA), was derived from the Lactococcus lactis peptidoglycan hydrolase AcmA. GEM particles were typically prepared from the innocuous bacterium L. lactis, and various parameters for the optimal preparation of GEM particles and binding of PA fusion proteins were determined. The versatility and flexibility of the display and delivery technology were demonstrated by investigating enzyme immobilization and nasal vaccine applications.

AcmA, the major autolysin of Lactococcus lactis MG1363 is a modular protein consisting of an N‐terminal active site domain and a C‐terminal peptidoglycan‐binding domain. The active site domain is homologous to that of muramidase‐2 of Enterococcus hirae, however, RP‐HPLC analysis of muropeptides released from Bacillus subtilis peptidoglycan, after digestion with AcmA, shows that AcmA is an N‐acetylglucosaminidase. In the C‐terminus of AcmA three highly similar repeated regions of 45 amino acid residues are present, which are separated by short nonhomologous sequences. The repeats of AcmA, which belong to the lysine motif (LysM) domain family, were consecutively deleted, removed, or, alternatively, one additional repeat was added, without destroying the cell wall‐hydrolyzing activity of the enzyme in vitro, although AcmA activity was reduced in all cases. In vivo, proteins containing no or only one repeat did not give rise to autolysis of lactococcal cells, whereas separation of the producer cells from the chains was incomplete. Exogenously added AcmA deletion derivatives carrying two repeats or four repeats bound to lactococcal cells, whereas the derivative with no or one repeat did not. In conclusion, these results show that AcmA needs three LysM domains for optimal peptidoglycan binding and biological functioning.

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AcmA, the major autolysin of Lactococcus lactis MG1363, is responsible for stationary phase cellular lysis and is involved in cell separation of this organism (9). The enzyme consists of two domains: the N-terminal region contains an N-acetyl-glucosaminidase active site domain (9; A. Steen, G. Buist, G. Horsburgh, S. J. Foster, O. P. Kuipers, and J. Kok, unpublished data) while the C-terminal region contains three so-called LysM domains, with which it specifically binds to peptidoglycan of L. lactis and of other gram-positive bacteria (49). Peptidoglycan, the major cell wall component in bacteria and the substrate of AcmA, consists of glycan strands cross-linked by peptide side chains. The peptide chain contains alternating L-and D-amino acids. D-Alanine (D-Ala) is incorporated into the peptidoglycan peptide moiety as a D-Ala-D-Ala dipeptide, where it is involved in cross-linking of adjacent peptidoglycan strands. In many bacteria alanine racemase is responsible for the synthesis of D-Ala from L-Ala, the naturally occurring alanine isomer (53). Bacillus subtilis expresses at least one alanine racemase: Dal (14). A dal mutant is dependent on D-Ala supplementation to be able to grow in a rich medium; cells start to lyse in the absence of D-Ala (4,14,20). In minimal medium the mutant is D-Ala dependent when L-Ala is supplemented, suggesting that a second, L-Ala-repressible racemase is present (4,14). Lactobacillus plantarum probably expresses only one alanine racemase, as an alr mutant is totally dependent on D-Ala for growth (22). D-Ala deprivation of an alr mutant of L. plantarum resulted in growth arrest, a rapid loss of cell viability, and an aberrant cell morphology (43). Electron microscopy analyses showed that mainly the cell septum is affected in this mutant. Like L. plantarum alr, L. lactis alr is totally dependent on the addition of D-Ala to the growth medium (23); when D-Ala was removed from the growth medium when the cells were in exponential growth phase, L. lactis alr growth was impaired and the culture started to lyse (17). The alr gene was used as a food-grade plasmid selection marker in the alr mutants of L. plantarum and L. lactis, complementing the D-Ala auxotrophy (7). Moreover, the alr mutants of L. plantarum and L. lactis were used in a mucosal vaccination study, in which these two mutants were shown to enhance the mucosal delivery of the tetanus toxin fragment C model antigen in mice (17).Although peptidoglycan covers the whole surface of L. lactis, AcmA binds to lactococcal cells at specific loci, namely around the poles and septum of the cell, exactly those places where cell lysis has been shown to start (35,49). Trichloroacetic acid treatment of cells causes binding of AcmA over the whole cell surface. Lipoteichoic acid (LTA) is a candidate-hindering component that is removed by this treatment, as it seems to be present in L. lactis at those positions where AcmA is not able to bind (49). LTA is a secondary cell wall polymer suggested to be involved in the control of autolysin activity (5, ...

Nuclear transport is facilitated by the Nuclear Pore Complex (NPC) and is essential for life in eukaryotes. The NPC is a long-lived and exceptionally large structure. We asked whether NPC quality control is compromised in aging mitotic cells. Our images of single yeast cells during aging, show that the abundance of several NPC components and NPC assembly factors decreases. Additionally, the single-cell life histories reveal that cells that better maintain those components are longer lived. The presence of herniations at the nuclear envelope of aged cells suggests that misassembled NPCs are accumulated in aged cells. Aged cells show decreased dynamics of transcription factor shuttling and increased nuclear compartmentalization. These functional changes are likely caused by the presence of misassembled NPCs, as we find that two NPC assembly mutants show similar transport phenotypes as aged cells. We conclude that NPC interphase assembly is a major challenge for aging mitotic cells.

Cellular aging is a multifactorial process that is characterized by a decline in homeostatic capacity, best described at the molecular level. Physicochemical properties such as pH and macromolecular crowding are essential to all molecular processes in cells and require maintenance. Whether a drift in physicochemical properties contributes to the overall decline of homeostasis in aging is not known. Here we show that the cytosol of yeast cells acidifies modestly in early aging and sharply after senescence. Using a macromolecular crowding sensor optimized for long-term FRET measurements, we show that crowding is rather stable and that the stability of crowding is a stronger predictor for lifespan than the absolute crowding levels. Additionally, in aged cells we observe drastic changes in organellar volume, leading to crowding on the µm scale, which we term organellar crowding. Our measurements provide an initial framework of physicochemical parameters of replicatively aged yeast cells.